Tony E. Godfrey

Exome and whole-genome sequencing of esophageal adenocarcinoma identifies recurrent driver events and mutational complexity

The incidence of esophageal adenocarcinoma (EAC) has risen 600% over the last 30 years. With a 5-year survival rate of ∼15%, the identification of new therapeutic targets for EAC is greatly important. We analyze the mutation spectra from whole-exome sequencing of 149 EAC tumor-normal pairs, 15 of which have also been subjected to whole-genome sequencing. We identify a mutational signature defined by a high prevalence of A>C transversions at AA dinucleotides. Statistical analysis of exome data identified 26 significantly mutated genes. Of these genes, five (TP53, CDKN2A, SMAD4, ARID1A and PIK3CA) have previously been implicated in EAC. The new significantly mutated genes include chromatin-modifying factors and candidate contributors SPG20, TLR4, ELMO1 and DOCK2. Functional analyses of EAC-derived mutations in ELMO1 identifies increased cellular invasion. Therefore, we suggest the potential activation of the RAC1 pathway as a contributor to EAC tumorigenesis.

MicroRNA expression profiles of esophageal cancer

OBJECTIVE Expression of microRNAs by array analysis provides unique profiles for classifying tissues and tumors. The purpose of our study was to examine microRNA expression in Barrett esophagus and esophageal cancer to identify potential markers for disease progression. METHODS MicroRNA was isolated from 35 frozen specimens (10 adenocarcinoma, 10 squamous cell carcinoma, 9 normal epithelium, 5 Barrett esophagus, and 1 high-grade dysplasia). MicroRNA expression was analyzed with Ambion bioarrays (Ambion, Austin, Tex) containing 328 human microRNA probes. RESULTS Unsupervised hierarchic clustering resulted in four major branches corresponding with four histologic groups. One branch consisted of 7 normal epithelium samples and 1 squamous cell carcinoma sample. The second branch consisted of 7 squamous cell carcinoma samples and 1 normal epithelium sample. The third branch contained 4 Barrett esophagus samples and 1 squamous cell carcinoma sample. The fourth contained all the adenocarcinoma samples and 1 sample each of Barrett esophagus, normal epithelium, squamous cell carcinoma, and high-grade dysplasia. Supervised classification with principal component analysis determined that the normal epithelium samples were more similar to the squamous cell carcinoma tumors, whereas the Barrett esophagus samples were more similar to adenocarcinoma. Pairwise comparisons between sample types revealed microRNAs that may be markers of tumor progression. Both mir_203 and mir_205 were expressed 2- to 10-fold lower in squamous cell carcinoma and adenocarcinomas than in normal epithelium. The mir_21 expression was 3- to 5-fold higher in both tumors than in normal epithelium. Prediction analysis of microarray classified 3 Barrett esophagus samples as Barrett esophagus, 1 as adenocarcinoma, and 1 as normal epithelium. CONCLUSION Expression profiles of miRNA distinguish esophageal tumor histology and can discriminate normal tissue from tumor. MicroRNA expression may prove useful for identifying patients with Barrett esophagus at high risk for progression to adenocarcinoma.

Gastrointestinal Adenocarcinomas of the Esophagus, Stomach, and Colon Exhibit Distinct Patterns of Genome Instability and Oncogenesis

A more detailed understanding of the somatic genetic events that drive gastrointestinal adenocarcinomas is necessary to improve diagnosis and therapy. Using data from high-density genomic profiling arrays, we conducted an analysis of somatic copy-number aberrations in 486 gastrointestinal adenocarcinomas including 296 esophageal and gastric cancers. Focal amplifications were substantially more prevalent in gastric/esophageal adenocarcinomas than colorectal tumors. We identified 64 regions of significant recurrent amplification and deletion, some shared and others unique to the adenocarcinoma types examined. Amplified genes were noted in 37% of gastric/esophageal tumors, including in therapeutically targetable kinases such as ERBB2, FGFR1, FGFR2, EGFR, and MET, suggesting the potential use of genomic amplifications as biomarkers to guide therapy of gastric and esophageal cancers where targeted therapeutics have been less developed compared with colorectal cancers. Amplified loci implicated genes with known involvement in carcinogenesis but also pointed to regions harboring potentially novel cancer genes, including a recurrent deletion found in 15% of esophageal tumors where the Runt transcription factor subunit RUNX1 was implicated, including by functional experiments in tissue culture. Together, our results defined genomic features that were common and distinct to various gut-derived adenocarcinomas, potentially informing novel opportunities for targeted therapeutic interventions.

Whole genome exon arrays identify differential expression of alternatively spliced, cancer-related genes in lung cancer

Alternative processing of pre-mRNA transcripts is a major source of protein diversity in eukaryotes and has been implicated in several disease processes including cancer. In this study we have performed a genome wide analysis of alternative splicing events in lung adenocarcinoma. We found that 2369 of the 17 800 core Refseq genes appear to have alternative transcripts that are differentially expressed in lung adenocarcinoma versus normal. According to their known functions the largest subset of these genes (30.8%) is believed to be cancer related. Detailed analysis was performed for several genes using PCR, quantitative RT-PCR and DNA sequencing. We found overexpression of ERG variant 2 but not variant 1 in lung tumors and overexpression of CEACAM1 variant 1 but not variant 2 in lung tumors but not in breast or colon tumors. We also identified a novel, overexpressed variant of CDH3 and verified the existence and overexpression of a novel variant of P16 transcribed from the CDKN2A locus. These findings demonstrate how analysis of alternative pre-mRNA processing can shed additional light on differences between tumors and normal tissues as well as between different tumor types. Such studies may lead to the development of additional tools for tumor diagnosis, prognosis and therapy.

Comparative Genomics of Esophageal Adenocarcinoma and Squamous Cell Carcinoma

BACKGROUND Esophageal cancer consists of two major histologic types: esophageal squamous cell carcinoma (ESCC), predominant globally, and esophageal adenocarcinoma (EAC), which has a higher incidence in westernized countries. Five-year overall survival is 15%. Clinical trials frequently combine histologic types although they are different diseases with distinct origins. In the evolving era of personalized medicine and targeted therapies, we hypothesized that ESCC and EAC have genomic differences important for developing new therapeutic strategies for esophageal cancer. METHODS We explored DNA copy number abnormalities in 70 ESCCs with publicly available array data and 189 EACs from our group. All data was from single nucleotide polymorphism arrays. Analysis was performed using a segmentation algorithm. Log ratio thresholds for copy number gain and loss were set at ±0.2 (approximately 2.3 and 1.7 copies, respectively). RESULTS The ESCC and EAC genomes showed some copy number abnormalities with similar frequencies (eg, CDKN2A, EGFR, KRAS, MYC, CDK6, MET) but also many copy number abnormalities with different frequencies between histologic types, most of which were amplification events. Some of these regions harbor genes for which targeted therapies are currently available (VEGFA, ERBB2) or for which agents are in clinical trials (PIK3CA, FGFR1). Other regions contain putative oncogenes that may be targeted in the future. CONCLUSIONS Using single nucleotide polymorphism arrays we compared genomic abnormalities in a large cohort of EACs and ESCCs. We report here the similar and different frequencies of copy number abnormalities in ESCC and EAC. These results may allow development of histology-specific therapeutic agents for esophageal cancer.

MicroRNA Prognostic Signature for Nodal Metastases and Survival in Esophageal Adenocarcinoma

BACKGROUND The incidence of esophageal adenocarcinoma is rapidly increasing and is now one of the leading causes of cancer death in the western world. MicroRNAs (miRNAs) are small noncoding RNAs that regulate the expression of protein-encoding genes and are involved in the development, progression and prognosis of other malignancies. We hypothesized that global miRNA expression would predict survival and lymph node involvement in a cohort of surgically resected esophagus cancer patients. METHODS The miRNA analysis was performed using a custom Affymetrix microarray with probes for 462 known human, 2,102 predicted human, 357 mouse, and 238 rat miRNAs. Expression of miRNA was evaluated in 45 primary tumors, and the association of miRNA expression with patient survival and lymph node metastasis was assessed. The prognostic impact of identified unique miRNAs was verified with quantitative reverse transcriptase polymerase chain reaction. RESULTS Our data indicate that the expression of individual human miRNA species is significantly associated with postresection patient survival. Using data from five unique miRNAs, we were further able to generate a combined miRNA expression signature that is associated with patient survival (p=0.005; hazard ratio 3.6) independent of node involvement and overall stage. The expression of three miRNAs (miR-99b and miR-199a_3p and _5p) was also associated with the presence of lymph node metastasis. CONCLUSIONS These results suggest miRNA expression profiling could provide prognostic utility in staging esophagus cancer patients and treatment planning with endoscopic and neoadjuvant therapies. The alterations of specific miRNAs may further elucidate steps in the metastatic pathway and allow for development of targeted therapy.

HER2 amplification, overexpression and score criteria in esophageal adenocarcinoma

The HER2 oncogene was recently reported to be amplified and overexpressed in esophageal adenocarcinoma. However, the relationship of HER2 amplification in esophageal adenocarcinoma with prognosis has not been well defined. The scoring systems for clinically evaluating HER2 in esophageal adenocarcinoma are not established. The aims of the study were to establish a HER2 scoring system and comprehensively investigate HER2 amplification and overexpression in esophageal adenocarcinoma and its precursor lesion. Using a tissue microarray, containing 116 cases of esophageal adenocarcinoma, 34 cases of Barretts esophagus, 18 cases of low-grade dysplasia and 15 cases of high-grade dysplasia, HER2 amplification and overexpression were analyzed by HercepTest and chromogenic in situ hybridization methods. The amplification frequency in an independent series of 116 esophageal adenocarcinoma samples was also analyzed using Affymetrix SNP 6.0 microarrays. In our studies, we have found that HER2 amplification does not associate with poor prognosis in total 232 esophageal adenocarcinoma patients by chromogenic in situ hybridization and high-density microarrays. We further confirm the similar frequency of HER2 amplification by chromogenic in situ hybridization (18%; 21 out of 116) and SNP 6.0 microarrays (16%, 19 out of 116) in esophageal adenocarcinoma. HER2 protein overexpression was observed in 12% (14 out of 116) of esophageal adenocarcinoma and 7% (1 out of 15) of high-grade dysplasia. No HER2 amplification or overexpression was identified in Barretts esophagus or low-grade dysplasia. All HER2 protein overexpression cases showed HER2 gene amplification. Gene amplification was found to be more frequent by chromogenic in situ hybridization than protein overexpression in esophageal adenocarcinoma (18 vs 12%). A modified two-step model for esophageal adenocarcinoma HER2 testing is recommended for clinical esophageal adenocarcinoma HER2 trial.

Intraoperative qRT-PCR for Detection of Lymph Node Metastasis in Head and Neck Cancer

Purpose: Sentinel node biopsy (SNB) has been shown to accurately stage the regional lymphatics in oral carcinoma. However, intraoperative pathology is only moderately sensitive and final pathology takes several days to complete. The purpose of this study was to develop a rapid, automated, and quantitative real-time PCR (qRT-PCR) assay that can match final pathology in an intraoperative time frame. Experimental Design: Four hundred forty-eight grossly tumor-negative lymph nodes were evaluated for expression of 3 markers [PVA (pemphigus vulgaris antigen), PTHrP (parathyroid hormone-related protein), and TACSTD1 (tumor-associated calcium signal transducer 1)]. Conformity of metastasis detection by qRT-PCR was determined using hematoxylin and eosin and immunohistochemistry staining as the gold standard. PVA and TACSTD1 were then multiplexed with β-glucuronidase to develop a rapid, automated single-tube qRT-PCR assay using the Cepheid GeneXpert system. This assay was used to analyze 103 lymph nodes in an intraoperative time frame. Results: Four hundred forty-two nodes produced an informative result for both qRT-PCR and pathologic examination. Concordance of qRT-PCR for individual markers with final pathology ranged from 93% to 98%. The best marker combination was TACSTD1 and PVA. A rapid, multiplex assay for TACSTD1 and PVA was developed on the Cepheid GeneXpert and demonstrated an excellent reproducibility and linearity. Analysis of 103 lymph nodes demonstrated 94.2% accuracy of this assay for identifying positive and negative nodes. The average time for each assay to yield results was 35 minutes. Conclusions: A rapid, automated qRT-PCR assay can detect lymph node metastasis in head and neck cancer with high accuracy compared to pathologic analysis and may be more accurate than intraoperative pathology. Combined, SNB and rapid qRT-PCR could more appropriately guide surgical treatment of patients with head and neck cancer. Clin Cancer Res; 17(7); 1858–66. ©2011 AACR.

Quantitative Expression and Immunogenicity of MAGE-3 and -6 in Upper Aerodigestive Tract Cancer

The MAGE antigens are frequently expressed cancer vaccine targets. However, quantitative analysis of MAGE expression in upper aerodigestive tract (UADT) tumor cells and its association with T‐cell recognition has not been performed, hindering the selection of appropriate candidates for MAGE‐specific immunotherapy. Using quantitative RT‐PCR (QRT‐PCR), we evaluated the expression of MAGE‐3/6 in 65 UADT cancers, 48 normal samples from tumor matched sites and 7 HLA‐A*0201+ squamous cell carcinoma of the head and neck (SCCHN) cell lines. Expression results were confirmed using Western blot. HLA‐A*0201:MAGE‐3‐ (271–279) specific cytotoxic T lymphocytes (MAGE‐CTL) from SCCHN patients and healthy donors showed that MAGE‐3/6 expression was highly associated with CTL recognition in vitro. On the basis of the MAGE‐3/6 expression, we could identify 31 (47%) of the 65 UADT tumors, which appeared to express MAGE‐3/6 at levels that correlated with efficient CTL recognition. To confirm that the level of MAGE‐3 expression was responsible for CTL recognition, 2 MAGE‐3/6 mRNAhigh SCCHN cell lines, PCI‐13 and PCI‐30, were subjected to MAGE‐3/6‐specific knockdown. RNAi‐transfected cells showed that MAGE expression and MAGE‐CTL recognition were significantly reduced. Furthermore, treatment of cells expressing low MAGE‐3/6 mRNA with a demethylating agent, 5‐aza‐2′‐deoxycytidine (DAC), increased the expression of MAGE‐3/6 and CTL recognition. Thus, using QRT‐PCR UADT cancers frequently express MAGE‐3/6 at levels sufficient for CTL recognition, supporting the use of a QRT‐PCR‐based assay for the selection of candidates likely to respond to MAGE‐3/6 immunotherapy. Demethylating agents could increase the number of patients amenable for targeting epigenetically modified tumor antigens in vaccine trials.

Rapid molecular detection of metastatic head and neck squamous cell carcinoma as an intraoperative adjunct to sentinel lymph node biopsy

Clinical staging of early head and neck squamous cell carcinoma (SCCHN) is often inaccurate, leading to elective neck dissection to detect the 30% of patients with micrometastatic disease. Sentinel node biopsy accurately stages the regional lymphatics, but intraoperative pathology is only moderately sensitive, and final pathology takes several days to complete. To facilitate immediate neck dissection where necessary, we have identified several promising marker genes of SCCHN metastasis and developed a rapid, accurate, and automated quantitative real‐time polymerase chain reaction (PCR) (qRT‐PCR) assay for intraoperative use.